The number, properties, and subunit composition of NMDA-type glutamate receptors (NMDA-Rs) present at a particular synapse must be under careful control in order to regulate calcium influx and different signaling cascades associated with the receptor and its activation. Synapses in many brain regions undergo a switch in the subunit composition of NMDA-Rs from predominantly containing NR2B to predominantly containing NR2A. This switch requires synaptic activity or sensory experience, alters receptor function, and it is thought to affect synaptic plasticity. Recent studies showed that NR2 subunits control the trafficking of NMDA-Rs into synapses with subunit-specific rules. NR2B is incorporated in a constitutive or activity- independent manner, whereas NR2A incorporation requires synaptic activity and is retained intracellularly in the absence of it. We propose to investigate the molecular determinants that control the differential trafficking of NMDA-Rs, the mechanisms involved in the switch of NR2 subunits, and the consequences of altering the composition of synaptic NMDA-Rs. The principal hypothesis is that NR2 subunits control synaptic insertion and stabilization of NMDA-Rs as well as synaptogenesis and spine morphology. These studies will use organotypic hippocampal slices from rat brain and several complementing methodologies, including molecular biology, biochemistry, fluorescence imaging, and electrophysiology. An innovative functional assay will be used to detect incorporation of NMDA-Rs into synapses. Chimeras of NR2 subunits will be used to examine the role played by different structural motifs. The specific aims are: 1) To test the hypothesis that NR2 subunits carry a signal that controls synaptic incorporation and stabilization of NMDA- Rs;2) To test the hypothesis that NR2A synaptic incorporation requires activity-dependent removal of NR2B-containing receptors and is independent of calcium influx;3) To test the hypothesis that NR2 subunits control the rate of synaptogenesis and shape the dynamics and morphology of dendritic spines. PUBLIC HEALTH RELEVANCE: Relevance NMDA-type glutamate receptors are a key component of synapses and they are involved in synaptic plasticity, brain development and pathology. The number, properties and subunit composition of the NMDA-Rs present at a particular synapse must be well controlled to regulate processes associated with the receptor and its activation. Our goal is to elucidate the molecular mechanisms that control the number and subunit composition of NMDA-Rs present at synapses. This field of research is poised to be of great significance for the understanding and treatment of multiple neuropsychiatric syndromes associated with cognitive deficits.